A non-surgical approach for male germ cell mediated gene transmission through transgenesis

Abstract

Microinjection of foreign DNA in male pronucleus by in-vitro embryo manipulation is difficult but remains the method of choice for generating transgenic animals. Other procedures, including retroviral and embryonic stem cell mediated transgenesis are equally complicated and have limitations. Although our previously reported technique of testicular transgenesis circumvented several limitations, it involved many steps, including surgery and hemicastration, which carried risk of infection and impotency. We improved this technique further, into a two step non-surgical electroporation procedure, for making transgenic mice. In this approach, transgene was delivered inside both testes by injection and modified parameters of electroporation were used for in-vivo gene integration in germ cells. Using variety of constructs, germ cell integration of the gene and its transmission in progeny was confirmed by PCR, slot blot and immunohistochemical analysis. This improved technique is efficient, requires substantially less time and can be easily adopted by various biomedical researchers.

Design, modelling and preliminary characterisation of microneedle-based electrodes for tissue electroporationin vivo

“…However, we did not observe GFP transgenics among one hundred offspring from mating testis-electroporated mice. One possible explanation is that we used the C57Bl/6 strain, which may not be optimal for this procedure, and so future work will involve testing the needle electrodes on mice from other genetic backgrounds [4]. Future work will also solve an issue of apparent heat damage to electrodes following a single use, by using different metallic coatings on electrodes.…”

“…This leads to a temporary increase in permeability of cell membranes, facilitating the passage of DNA to cells. DNA injection into testis, followed by electroporation, has been reported as a novel method of producing transgenic rodent offspring [3,4]. The ability to produce transgenic rodents by testis electroporation would greatly reduce the costs and skill sets needed to produce this essential resource required for the study of developmental biology and disease pathogenesis.…”

Production of Transgenic Rats

“…Moreover, many hundreds of peer-reviewed papers describing useful results from the genetic modification of rat models of human disease are published every year. New methods of transgenesis are being developed such as testicular tissue electroporation that if validated have the potential to greatly simplify the production of transgenic rats (Usmani, et al, 2013). Using embryonic stem cells to produce gene knockouts in the rat is now possible and the dream of knockouts in genes of choice has been realized with completely new technologies that allow genome editing and the generation of allelic series.…”

An Efficient Method for Generation of Transgenic Rats Avoiding Embryo Manipulation

“…11 The sperm were isolated from the epididymis of these fore founder and were analyzed by a flow cytometer for EGFP expression (FACS caliber, BD Biosciences, San Jose, CA). For immuohistochemical localization of EGFP, the testes of electroporated rats were isolated after 15 days of electroporation and fixed in 4% paraformaldehyde for 20 hours before they were processed and embedded in paraffin.…”